Cloning And Expression Analysis Of TLR Signaling Pathway Related Genes Of Apostichopus Japonicus

In recent years,the demand for Apostichopus japonicus has been increasing.Although it drives the development of the culture industry,it also brings disease problems and causes great economic losses.Therefore,we hope to solve the disease problems fundamentally by improving the immunity of the A.japonicus.In this study,we cloned and analyzed genes related to TLR signaling pathway in A.japonicus,and provided theoretical basis for immune-related research in A.japonicus.1.A gene SARM in the TLR signal pathway of A.japonicus was cloned by RACE which was named AjSARM.The AjSARM gene has a total length of 3874 bp and an open reading frame of 2412 bp,encoding 803 amino acids.By applying SMART software to predict the protein structure of AjSARM,it was found that this protein consists of two SAM domains and one TIR domain at the c-terminal.At the same time,the real-time fluorescence quantitative PCR method was used to measure the expression of AjSARM in different development stages of A.japonicus,and the results showed that the highest expression was in the five tentacles of A.japonicus.2.Aestivation is an indispensable period for A.japonicus.During aestivation,A.japonicus will produce a series of physiological and morphological changes.In this paper,12 genes of two pathways(Myd88-dependent pathway and Myd88-independent pathway)in the TLR signal pathway of A.japonicus were measured by real-time fluorescence quantitative PCR in five different periods under temperature challenge(15 before aestivation,22 before aestivation,28 during aestivation,22 after aestivation recovery,15 after aestivation recovery)and five different tissues(coelomocyte,body wall,respiratory tree,tube feet and intestine)The results showed that all genes in the body wall and coelomic cells were down-regulated at four time points compared with the control group.In intestinal tissues,the expression levels of 12 genes were up-regulated at 22℃ before aestivation,down-regulated at 28℃,up-regulated at 22℃ during aestivation recovery,and down-regulated at 15℃.In the respiratory tree and tube feet,the change trend of the other 11 genes was the same except that the TRAF3 gene expression was down-regulated in tube feet at 28℃ in aestivation.3.The conserved domains in the amino acid sequences of Toll、MyD88、IRAK4、TRAF6、P105 and Rel in the dependent pathway of A.japonicus TLR signal pathway MyD88 were compared and analyzed to find possible interaction sites between upstream and downstream genes.The expression patterns of these six genes in coelomocytes at four time points after Vibrio splendidus stimulation were detected by real-time fluorescence quantitative PCR.The results showed that Toll gene expression was slightly up-regulated at 12 h after receiving immune stimulation,and was inhibited at other time points.The expression patterns of MyD88 and TRAF6 are basically the same,with the highest expression level at 12 h.IRAK4 expression was up-regulated at 12 h and returned to the initial state at 48 h.P105 and Rel are two homologs of NFκB Kappa B,which have similar domains,and the expression patterns of the two genes are consistent.A highly conserved TLR signal pathway was found in A.japonicus through comparative analysis of gene sequences and expression patterns.In response to pathogen invasion,the six genes in this pathway cooperate and show significant positive correlation,participating in signal transduction and immune response together.